The primary goal of this proposal is to develop and finalize configuration of a rapid molecular diagnostics test to predict antibiotic resistance of th major human uropathogens - Escherichia coli, Klebsiella pneumoniae, Enterobacter spp and P. mirabilis - by determining the sub-species clonal identity (clonotype) of the infecting bacteria directly from urine samples. Currently, the empirical (pre- antibiogram) choice of antibiotic treatment is guided by an educated guess about the suspected species of pathogen and its cumulative antibiogram from data compiled locally every year. We have shown, in the Phase I, studies that a significant improvement in antibiotic choice against uropathogenic E. coli could be achieved if the antibiotic treatment choice would be guided by cumulative antibiogram of the clonotype to which the infecting E. coli strain belongs. We also have shown that the clonal identity of E. coli can be determined directly in urine using qPCR. The basic idea for the Phase II studies is to refine the E. coli clonotyping test and add to the test clonal and/or species markers for non- E. coli uropathogens, with a goal of covering 90-95% of pathogens, and reduce the potential drug-bug mismatch for common antibiotics at least 3-folg. Clonotype determination is done by detection of the presence/absence of a limited number (e.g. six to ten) of polymorphic gene markers - mosaically- distributed genes and/or nucleotide polymorphisms - that will be selected genome-wide. Interrogation of the markers will be performed by qPCR using a simple, adaptable configuration of standard PCR tube strips and existing FDA-cleared instrumentation platforms. To achieve these goals, we will determine genome sequences and cumulative antibiograms of a comprehensive number of E. coli and non-E. coli clinical isolates, and establish the association between the resistance to common antibiotics and clonal identity of the pathogens. Polymorphic clonal markers, that are suitable for binary differentiation, will be identified across the genome of each species. Oligonucleotide primers, capable of robust interrogation of the binary loci, will be designed and validated using clinical isolates from different geographic regions of the USA over 3 years. The proposed studies will be performed as a collaboration between ID Genomics, Inc., a Seattle-based start-up company, specializing in sequence-based typing of microbial pathogens, and the University of Washington, Seattle, the laboratory of Evgeni Sokurenko, and a consortium of eight different clinical microbiology laboratories.